Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI, 48109, USA.
Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI, 48109, USA; Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, 48109, USA; Department of Cell & Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, 48109, USA.
Biomaterials. 2021 Aug;275:120898. doi: 10.1016/j.biomaterials.2021.120898. Epub 2021 May 18.
Human embryonic stem cells (hESCs) have the intrinsic capacity to self-organize and generate patterned tissues. In vitro models that coax hESCs to form embryonic-like structures by modulating physical environments and priming with chemical signals have become a powerful tool for dissecting the regulatory mechanisms underlying early human development. Here we present a 3D suspension culture system of hESCs that can generate post-implantation, pre-gastrulation embryonic-like tissues in an efficient and controllable manner. The efficiency of the development of asymmetric tissues, which mimic the post-implantation, pre-gastrulation amniotic sac, was about 50% in the 3D suspension culture. Quantitative imaging profiling and unsupervised trajectory analysis revealed that hESC aggregates first entered into a transitional stage expressing Brachyury (or T), before their development branched into different paths to develop into asymmetric embryonic-like tissues, amniotic-like tissues, and mesodermal-like tissues, respectively. Moreover, the branching developmental trajectory of embryonic-like structures was affected by the initial cell seeding density or cluster size of hESCs. A higher percentage of amniotic-like tissues was observed under a small initial cell seeding density of hESCs. Conversely, a large initial cell seeding density of hESCs promoted the development of mesodermal-like tissues. Intermediate cell seeding densities of hESCs in the 3D suspension culture promoted the development of asymmetric embryonic-like tissues. Our results suggest that hESCs have the intrinsic capability to sense the initial cell population size, which in turn regulates their differentiation and self-organization into different embryonic-like tissues. Our 3D suspension culture thus provides a promising experimental tool to study the interplay between tissue topology and self-organization and progressive embryonic development using in vitro hESC-based models.
人类胚胎干细胞（hESCs）具有内在的自我组织和产生模式化组织的能力。通过调节物理环境和化学信号诱导，将 hESCs 诱导形成胚胎样结构的体外模型已成为解析早期人类发育背后调控机制的有力工具。在这里，我们提出了一种 hESC 的 3D 悬浮培养系统，该系统能够以高效和可控的方式生成着床后、原肠胚前的胚胎样组织。在 3D 悬浮培养中，模拟着床后、原肠胚前羊膜囊的不对称组织的发育效率约为 50%。定量成像分析和无监督轨迹分析显示，hESC 聚集物首先进入表达 Brachyury（或 T）的过渡阶段，然后其发育分支为不同的路径，分别发育成不对称的胚胎样组织、羊膜样组织和中胚层样组织。此外，胚胎样结构的分支发育轨迹受 hESC 初始细胞接种密度或细胞簇大小的影响。在 hESC 初始细胞接种密度较低的情况下，观察到更多的羊膜样组织。相反，高初始细胞接种密度促进中胚层样组织的发育。hESC 在 3D 悬浮培养中的中等初始细胞接种密度促进了不对称胚胎样组织的发育。我们的研究结果表明，hESC 具有内在的能力来感知初始细胞群体大小，进而调节其分化和自我组织为不同的胚胎样组织。我们的 3D 悬浮培养因此为使用基于 hESC 的体外模型研究组织拓扑和自我组织与胚胎发育之间的相互作用提供了一个有前途的实验工具。
